Lettuce variety 81-296 RZ

ABSTRACT

The present invention relates to a Lactuca sativa seed designated 81-296 RZ. The present invention also relates to a Lactuca sativa plant produced by growing the 81-296 RZ seed. The invention further relates to methods for producing the lettuce cultivar, represented by lettuce variety 81-296 RZ.

APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional patent applicationSer. No. 62/490,708 filed Apr. 27, 2017.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a new lettuce (Lactuca sativa) varietydesignated 81-296 RZ. Lettuce cultivar 81-296 RZ exhibits a combinationof traits including resistance to downy mildew (Bremia lactucae) racesBl:1 to Bl:32 and Ca-I to Ca-VIII, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, resistance to Lettuce Mosaic Virus(LMV) race LMV:1, as well as having thick leaves, white colored seedsand a reduced wound-induced surface discoloration of the leaves.

BACKGROUND OF THE INVENTION

All cultivated forms of lettuce belong to the highly polymorphicspecies, Lactuca sativa, which is grown for its edible head and leaves.As a crop, lettuces are grown commercially wherever environmentalconditions permit the production of an economically viable yield.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae)family. Lettuce is related to chicory, sunflower, aster, scorzonera,dandelion, artichoke and chrysanthemum. Sativa is one of about 300species in the genus Lactuca.

Lettuce cultivars are susceptible to a number of pests and diseases suchas downy mildew (Bremia lactucae). Every year this disease leads tomillions of dollars of lost lettuce crop throughout the world. Downymildew (Bremia lactucae) is highly destructive on lettuce grown atrelatively low temperature and high humidity. Downy mildew is caused bya fungus, Bremia lactucae, which can be one of the following strains:NL1, NL2, NL4, NL5, NL6, NL7, NL10, NL12, NL13, NL14, NL15, NL16, Bl:17,Bl:18, Bl:20, Bl:21, Bl:22, Bl:23, Bl:24, Bl:25, Bl:26, Bl:27, Bl:28,Bl:29, Bl:30, Bl:31, Bl:32 (Van Ettekoven, K. et al., “Identificationand denomination of ‘new’ races of Bremia lactucae,” In: Lebeda, A. andKristkova, E (eds.), Eucarpia Leafy Vegetables, 1999, PalackyUniversity, Olomouc, Czech Republic, pp. 171-175; Van der Arend et al.“Identification and denomination of “new” races of Bremia lactucae inEurope by IBEB until 2002.” In: Van Hintum, Th et al. (eds.), EucarpiaLeafy Vegetables Conference 2003, Centre for Genetic Resources,Wageningen, The Netherlands, p. 151; Plantum NL (Dutch association forbreeding, tissue culture, production and trade of seeds and youngplants), Van der Arend et al. “Identification and denomination of “new”races of Bremia lactucae in Europe by IBEB until 2002.” In: Van Hintum,Th et al. (eds.), Eucarpia Leafy Vegetables Conference 2003, Centre forGenetic Resources, Wageningen, The Netherlands, p. 151; Plantum NL(Dutch association for breeding, tissue culture, production and trade ofseeds and young plants); IBEB press release “New race of Bremia lactucaeBl:27 identified and nominated”, May 2010; Plantum NL (Dutch associationfor breeding, tissue culture, production and trade of seeds and youngplants), “New race of Bremia lactucae Bl:28 identified and nominated”,March 2011; Plantum NL (Dutch association for breeding, tissue culture,production and trade of seeds and young plants), IBEB press release,“New races of Bremia lactucae, Bl:29, Bl:30 and Bl:31 identified andnominated”, August 2013; Plantum NL (Dutch association for breeding,tissue culture, production and trade of seeds and young plants), IBEBpress release, “A new race of Bremia lactucae, Bl:32 identified andnominated in Europe”, May 2015), Ca-I, Ca-IIA, Ca-IIB, Ca-III, Ca-IV(Schettini, T. M., Legg, E. J., Michelmore, R. W., 1991. Insensitivityto metalaxyl in California populations of Bremia lactucae and resistanceof California lettuce cultivars to downy mildew. Phytopathology 81(1).p. 64-70), and Ca-V, Ca-VI, Ca-VII, Ca-VIII (Michelmore R. & Ochoa. O.“Breeding Crisphead Lettuce.” In: California Lettuce Research Board,Annual Report 2005-2006, 2006, Salinas, Calif., pp. 55-68).

Downy mildew causes pale, angular, yellow areas bounded by veins on theupper leaf surfaces. Sporulation occurs on the opposite surface of theleaves. The lesions eventually turn brown, and they may enlarge andcoalesce. These symptoms typically occur first on the lower leaves ofthe lettuce, but under ideal conditions may move into the upper leavesof the head. When the fungus progresses to this degree, the head cannotbe harvested. Less severe damage requires the removal of more leavesthan usual, especially when the lettuce reaches its final destination.

Of the various species of aphids that feed on lettuce, thecurrant-lettuce aphid (Nasonovia ribisnigri) is the most destructivespecies because it feeds both on the leaves of the lettuce as well asthe heart of the lettuce, making it difficult to control withconventional insecticides. The lettuce aphid feeds by sucking sap fromthe lettuce leaves. Although direct damage to the lettuce may belimited, its infestation has serious consequences because the presenceof aphids makes lettuce unacceptable to consumers.

Lettuce mosaic virus (LMV) mainly infects lettuce seeds, which is theprimary way that the virus is introduced to lettuce in the fields, butalso can infect numerous crops and weeds, thereby creating reservoirs ofthe virus. LMV also can be vectored by aphids, which spread the viruswithin a lettuce field and introduce it into lettuce fields frominfected weeds and crops outside the field.

Symptoms of LMV vary greatly. Leaves of plants that are infected at ayoung stage are stunted, deformed and (in some varieties) show a mosaicor mottling pattern. Such plants rarely grow to full size; head lettucevarieties infected early fail to form heads. Plants that are infectedlater in the growth cycle show a different set of symptoms. These plantsmay reach full size, but the older outer leaves turn yellow, twisted,and otherwise are deformed. On head lettuce, the wrapper leaves oftenwill curve back away from the head and developing heads may be deformed.In some cases, brown necrotic flecks occur on the wrapper leaves.

The Leaf lettuce type is appreciated by consumers for its crisp,pleasant tasting leaves. In Europe, this type of lettuce is oftenreferred to as Batavia lettuce. Leaf lettuces grow upright, with looseleaves and are non-heading.

The production of packaged salad mixes, for example, involves harvestingand processing of lettuce, which induces a strong wound response on thecut surfaces. Such a wound-induced response can lead to a rapiddeterioration of the processed product. This deterioration is manifestedas a brown or pink discoloration at, or adjacent to the wound surface.The brown or pink color that develops over time on the cut leaf surfacemakes the product highly unattractive to potential customers.

The wound-induced surface discoloration can in part be countered byreducing oxygen levels in the packaging. However, using specializedpackaging is costly and may result in anaerobic conditions. Anaerobicconditions may reduce shelf life and may promote the growth ofmicro-organisms that produce a bad smell, reducing the overallattractiveness and taste of the product. Therefore lettuce plants thatexhibit reduced wound-induced discoloration of cut leaf surfaces isdesirable. Such lettuce plants stay visually more attractivepost-processing, and the need for reduced oxygen levels in the packagingof the cut leaves of these lettuce plants is minimized.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

Given the need expressed by relevant stakeholders for a lettuce varietywhich is resistant to downy mildew (Bremia lactucae) races Bl:1 to Bl:32and Ca-I to Ca-VIII, resistant to currant-lettuce aphid (Nasonoviaribisnigri) biotype Nr:0, resistant to Lettuce Mosaic Virus (LMV) raceLMV:1, has thick leaves, white colored seeds and a reduced wound-inducedsurface discoloration of the leaves, the present invention addressesthis need by providing a new type of lettuce (Lactuca sativa) varietydesignated 81-296 RZ.

The present invention provides a new lettuce variety designated 81-296RZ. This new lettuce variety exhibits a combination of traits includingresistance to downy mildew (Bremia lactucae) races Bl:1 to Bl:32 andCa-I to Ca-VIII, resistance to currant-lettuce aphid (Nasonoviaribisnigri) biotype Nr:0, resistance to Lettuce Mosaic Virus (LMV) raceLMV:1, as well as having thick leaves, white colored seeds and a reducedwound-induced surface discoloration of the leaves.

The present invention provides seed of a lettuce (Lactuca sativa)variety designated 81-296 RZ. A sample of seeds of said lettuce variety,have been deposited with the National Collections of Industrial, Marineand Food Bacteria (NCIMB) in Bucksburn, Aberdeen AB21 9YA, Scotland, UKand have been assigned NCIMB Accession No. 42738.

In one embodiment, the invention provides a lettuce plant grown from theseed of lettuce (Lactuca sativa) variety 81-296 RZ.

In another embodiment, the invention provides a lettuce plant designated81-296 RZ, which is a plant grown from seed having been deposited underNCIMB Accession No. 42738.

In one embodiment, the invention provides for a lettuce plant which maycomprise genetic information for exhibiting all of the physiological andmorphological characteristics of a plant of the invention, wherein thegenetic information is as contained in a plant, a sample of seed of saidvariety having been deposited under NCIMB Accession No. 42738.

In one embodiment, the invention provides for a lettuce plant exhibitingall the physiological and morphological characteristics of a plant ofthe invention, and having the genetic information for so exhibiting thecombination of traits, wherein the genetic information is as containedin a plant, a sample of seed of said variety having been deposited underNCIMB Accession No. 42738.

In an embodiment of the present invention, there also is provided partsof a lettuce plant of the invention, which may include parts of alettuce plant exhibiting all the physiological and morphologicalcharacteristics of a plant of the invention, or parts of a lettuce planthaving any of the mentioned resistance(s) and a combination of traitsincluding one or more or all morphological and physiologicalcharacteristics tabulated herein, including parts of lettuce variety81-296 RZ, wherein the plant parts are involved in sexual reproduction,which include, without limitation, microspores, pollen, ovaries, ovules,embryo sacs or egg cells and/or wherein the plant parts are suitable forvegetative reproduction, which include, without limitation, cuttings,roots, stems, cells or protoplasts and/or wherein the plant parts aretissue culture of regenerable cells in which the cells or protoplasts ofthe tissue culture are derived from a tissue such as, for example andwithout limitation, leaves, pollen, embryos, cotyledon, hypocotyls,meristematic cells, roots, root tips, anthers, flowers, seeds or stems.The plants of the invention from which such parts may come from includethose wherein a sample of seed of which having been deposited underNCIMB Accession No. 42738 or lettuce variety or cultivar designated81-296 RZ, as well as seed from such a plant, plant parts of such aplant (such as those mentioned herein) and plants from such seed and/orprogeny of such a plant, advantageously progeny exhibiting suchcombination of such traits, each of which, is within the scope of theinvention; and such combination of traits.

In a further embodiment there is a plant regenerated from theabove-described plant parts or regenerated from the above-describedtissue culture. Advantageously such a plant may have morphologicaland/or physiological characteristics of lettuce variety 81-296 RZ and/orof a plant grown from seed, a sample of seed of which having beendeposited under NCIMB Accession No. 42738—including without limitationsuch plants having all of the physiological and morphologicalcharacteristics of lettuce variety 81-296 RZ and/or of a plant grownfrom seed, a sample of seed of which having been deposited under NCIMBAccession No. 42738.

Accordingly, in still a further embodiment, there is provided a lettuceplant having all of the physiological and morphological characteristicsof lettuce variety 81-296 RZ, a sample of seed of which having beendeposited under NCIMB Accession No. 42738. Such a plant may be grownfrom the seeds, regenerated from the above-described plant parts, orregenerated from the above-described tissue culture. A lettuce planthaving any of the aforementioned resistance(s), and one or moremorphological or physiological characteristics recited or tabulatedherein, and a lettuce plant advantageously having all of theaforementioned resistances and the characteristics recited and tabulatedherein, are preferred. Parts of such plants—such as those plant partsabove-mentioned—are encompassed by the invention.

In a further aspect, the invention provides a method of vegetativelypropagating a plant of lettuce variety 81-296 RZ which may comprise (a)collecting tissue capable of being propagated from a plant of lettuce81-296 RZ, a sample of seed of said variety having been deposited underNCIMB accession No. 42738 and (b) cultivating the tissue to obtainproliferated shoots and rooting the proliferated shoots to obtain rootedplantlets. Optionally the invention further may comprise growing plantsfrom the rooted plantlets. Plantlets and plants produced by thesemethods, are encompassed by the invention. In one embodiment, there isprovided a method for producing a progeny of lettuce cultivar 81-296 RZwhich may comprise crossing a plant designated 81-296 RZ with itself orwith another lettuce plant, harvesting the resultant seed, and growingsaid seed.

In a further embodiment, a progeny plant is provided which is producedby this method, wherein said progeny exhibits a combination of traitsincluding resistance to downy mildew (Bremia lactucae) races Bl:1 toBl:32 and Ca-I to Ca-VIII, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, resistance to Lettuce Mosaic Virus(LMV) race LMV:1, as well as having thick leaves, white colored seedsand a reduced wound-induced surface discoloration of the leaves.

In another embodiment, a progeny plant is provided which is produced bythe above method, wherein said progeny exhibits all the morphologicaland physiological characteristics of the lettuce variety designated81-296 RZ, a sample of seed of said variety having been deposited underNCIMB Accession No. 42738.

In a further embodiment there is provided a progeny plant produced bysexual or vegetative reproduction, grown from seeds, regenerated fromthe above-described plant parts, or regenerated from the above-describedtissue culture of the lettuce cultivar or a progeny plant thereof, asample of seed of which having been deposited under NCIMB Accession No.42738. The progeny may have any of the aforementioned resistance(s), andone or more morphological or physiological characteristics recited ortabulated herein, and a progeny plant advantageously having all of theaforementioned resistances and the characteristics recited and tabulatedherein, are preferred.

Progeny of the lettuce variety 81-296 RZ may be modified in one or moreother characteristics, in which the modification is a result of, forexample and without limitation, mutagenesis or transformation with atransgene.

In still another embodiment, the present invention provides progeny oflettuce cultivar 81-296 RZ produced by sexual or vegetativereproduction, grown from seed, regenerated from the above-describedplant parts, or regenerated from the above-described tissue culture ofthe lettuce cultivar or a progeny plant thereof.

The invention further relates to a method for producing a seed of a81-296 RZ-derived lettuce plant which may comprise (a) crossing a plantof lettuce variety 81-296 RZ, a sample of seed of which having beendeposited under NCIMB Accession No. 42738, with a second lettuce plant,and (b) whereby seed of a 81-296 RZ-derived lettuce plant forms. Thismethod may further comprise (c) crossing a plant grown from 81-296RZ-derived lettuce seed with itself or with a second lettuce plant toyield additional 81-296 RZ-derived lettuce seed, (d) growing theadditional 81-296 RZ-derived lettuce seed of step (c) to yieldadditional 81-296 RZ-derived lettuce plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to generate further 81-296 RZ-derived lettuce plants, and(f) whereby seed of a 81-296 RZ-derived lettuce plant forms. A seedproduced by this method and a plant grown from said seed also form partof the invention.

The invention also relates to a method of introducing at least one newtrait into a plant of lettuce variety 81-296 RZ which may comprise: (a)crossing a plant of lettuce variety 81-296 RZ, a sample of seed of whichhaving been deposited under NCIMB Accession No. 42738, with a secondlettuce plant that may comprise at least one new trait to produceprogeny seed, (b) harvesting and planting the progeny seed to produce atleast one progeny plant of a subsequent generation, wherein the progenyplant may comprise the at least one new trait, (c) crossing the progenyplant with a plant of lettuce variety 81-296 RZ to produce backcrossprogeny seed, (d) harvesting and planting the backcross progeny seed toproduce a backcross progeny plant, and (e) repeating steps (c) and (d)for at least three additional generations to produce a lettuce plant ofvariety 81-296 RZ which may comprise at least one new trait and all ofthe physiological and morphological characteristics of a plant oflettuce variety 81-296 RZ, when grown in the same environmentalconditions. A lettuce plant produced by this method also forms part ofthe invention.

The invention further relates to a method of producing a plant oflettuce variety 81-296 RZ which may comprise at least one new trait, themethod which may comprise introducing a mutation or transgene conferringthe at least one new trait into a plant of lettuce variety 81-296 RZ,wherein a sample of seed of said variety has been deposited under NCIMBAccession No. 42738. A lettuce plant produced by this method also formspart of the invention.

In still a further embodiment, the invention provides a method ofproducing a lettuce seed which may comprise crossing a male parentlettuce plant with a female parent lettuce plant and harvesting theresultant lettuce seed, in which the male parent lettuce plant or thefemale parent lettuce plant is a lettuce plant of the invention, e.g. alettuce plant having all of the morphological or physiologicalcharacteristics tabulated herein, including a lettuce plant of lettucecultivar 81-296 RZ, a sample of seed of which having been depositedunder 42738. The resultant lettuce seed produced by this method and thelettuce plant that is produced by growing said lettuce seed also formspart of the invention.

In still a further embodiment, the invention provides a method ofproducing a lettuce cultivar which exhibits all of the physiological andmorphological characteristics of lettuce variety 81-296 RZ, a sample ofseed of said variety having been deposited under NCIMB accession No.42738.

The invention even further relates to a method of producing lettuceleaves as a food product which may comprise: (a) sowing a seed oflettuce variety 81-296 RZ, a sample of seed of which having beendeposited under NCIMB Accession No. 42738, (b) growing said seed into aharvestable lettuce plant and (c) harvesting lettuce leaves or headsfrom the plant. The invention further comprehends packaging and/orprocessing the lettuce plants, heads or leaves.

Also encompassed by the invention is a container which may comprise oneor more lettuce plants of the invention for harvest of leaves.

Further encompassed by the invention is a method of determining thegenotype of a plant of lettuce variety 81-296 RZ, a sample of seed ofwhich has been deposited under NCIMB Accession No. 42738, or a firstgeneration progeny thereof, which may comprise obtaining a sample ofnucleic acids from said plant and comparing said nucleic acids to asample of nucleic acids obtained from a reference plant, and detecting aplurality of polymorphisms between the two nucleic acid samples, whereinthe plurality of polymorphisms are indicative of lettuce variety 81-296RZ and/or give rise to the expression of any one or more, or all, of thephysiological and morphological characteristics of lettuce variety81-296 RZ of the invention.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph), such that Applicants reservethe right and hereby disclose a disclaimer of any previously describedproduct, process of making the product, or method of using the product.

It is noted that in this disclosure and particularly in the claims,terms such as “comprises”, “comprised”, and “comprising” and the like(e.g., “includes”, “included”, “including”, “contains”, “contained”,“containing”, “has”, “had”, “having”, etc.) can have the meaningascribed to them in US Patent law, i.e., they are open ended terms. Forexample, any method that “comprises,” “has” or “includes” one or moresteps is not limited to possessing only those one or more steps and alsocovers other unlisted steps. Similarly, any plant that “comprises,”“has” or “includes” one or more traits is not limited to possessing onlythose one or more traits and covers other unlisted traits. Similarly,the terms “consists essentially of” and “consisting essentially of” havethe meaning ascribed to them in US Patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention. See also MPEP § 2111.03. In addition, the term “about” isused to indicate that a value includes the standard deviation of errorfor the device or method being employed to determine the value.

These and other embodiments are disclosed or are obvious from andencompassed by the following Detailed Description.

DEPOSIT

The Deposit with NCIMB Ltd, Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen AB21 9YA, UK, on Apr. 18, 2017, under depositaccession number 42738 was made pursuant to the terms of the BudapestTreaty. Upon issuance of a patent, all restrictions upon the depositwill be removed, and the deposit is intended to meet the requirements of37 CFR §§ 1.801-1.809. The deposit will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent and for the enforceable life of the patent. The deposit will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced if necessary during that period.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawing, in which:

FIG. 1 is an illustration of six different shapes of the fourth leaffrom a 20-day old seedling grown under optimal conditions.

FIG. 2 is a picture of the shape of the fourth leaf of 81-296 RZ.

FIG. 3 is a picture of the shape of a mature leaf of 81-296 RZ.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of a new lettuce variety herein referred to aslettuce variety 81-296 RZ. Lettuce variety 81-296 RZ is a uniform andstable line, distinct from other such lines.

In a preferred embodiment, the specific type of breeding method employedfor developing a lettuce cultivar is pedigree selection, where bothsingle plant selection and mass selection practices are employed.Pedigree selection, also known as the “Vilmorin system of selection,” isdescribed in Fehr, W., Principles of Cultivar Development, Volume I,MacMillan Publishing Co., which is hereby incorporated by reference.

When pedigree selection is applied, in general selection is firstpracticed among F₂ plants. In the next season, the most desirable F₃lines are first identified, and then desirable F₃ plants within eachline are selected. The following season and in all subsequentgenerations of inbreeding, the most desirable families are identifiedfirst, then desirable lines within the selected families are chosen, andfinally desirable plants within selected lines are harvestedindividually. A family refers to lines that were derived from plantsselected from the same progeny from the preceding generation.

Using this pedigree method, two parents may be crossed using anemasculated female and a pollen donor (male) to produce F₁ offspring.Lettuce is an obligate self-pollination species, which means that pollenis shed before stigma emergence, assuring 100% self-fertilization.Therefore, in order to optimize crossing, a method of misting may beused to wash the pollen off prior to fertilization to assure crossing orhybridization.

Parental varieties are selected from commercial varieties thatindividually exhibit one or more desired phenotypes. Additionally, anybreeding method involving selection of plants for the desired phenotypemay be used in the method of the present invention.

The F₁ may be self-pollinated to produce a segregating F₂ generation.Individual plants may then be selected which represent the desiredphenotype in each generation (F₃, F₄, F₅, etc.) until the traits arehomozygous or fixed within a breeding population.

A detailed description of the development of lettuce variety 81-296 RZis described in Table 1. The seedlot in year 3-4, seedlot 16R.6304, wasdeposited with the NCIMB under deposit number 42738.

TABLE 1 Year Description Location 0 Final F1-cross 09B.250989 × plant12F.40035 Fijnaart, in glass house. The Netherlands 0 F1 plant grown forF2 seed production in glass Fijnaart, house. The Netherlands 1 F2 plantselected in open field, followed by F3 Soret, France seed production. 2F3 plant selected in plastic tunnel, followed by Aramon, France F4 seedproduction. 2 F4 plant selected in open field, followed by F5 Aramon,France seed production. 3 F5 plant selected in open field, followed byF6 Fijnaart, seed production The Netherlands 3-4 F6 line establisheduniform, multiplied in Daylesford, plastic tunnel: seed lot 16R.6304Australia

In one embodiment, a plant of the invention has all the physiologicaland morphological characteristics of lettuce variety 81-296 RZ. Thesecharacteristics of a lettuce plant of the invention, e.g. variety 81-296RZ, are summarized in Tables 2-4.

Next to the physiological and morphological characteristics mentioned inTables 2-4, a plant of the invention also exhibits resistance to downymildew (Bremia lactucae Regel.) races Bl: 1-32 and Ca-I to Ca-VIII.

As used herein resistance against Bremia lactucae is defined as theability of a plant to resist infection by each of the various strainsBl:1 to Bl:32, Ca-I to Ca-VIII of Bremia lactucae Regel. in all stagesbetween the seedling stage and the harvestable plant stage. Bl:1 toBl:32 means strains NL1, NL2, NL4, NL5, NL6, NL7, NL10, NL12, NL13,NL14, NL15, NL16, Bl:17, Bl:18, Bl:20, Bl:21, Bl:22, Bl:23, Bl:24,Bl:25, Bl:26, Bl:27, Bl:28, Bl:29, Bl:30, Bl:31, Bl:32 (Van Ettekoven K,Van der Arend A J M, 1999. identification and denomination of ‘new’races of Bremia lactucae. In: Lebeda A, Kristkova E (eds.) Eucarpialeafy vegetables '99. Palacky University, Olomouc, Czech Republic, 1999:171-175; Van der Arend, A. J. M., Gautier, J., Guenard, M., Michel, H.,Moreau, B., de Ruijter, J., Schut, J. W. and de Witte, I. (2003).Identification and denomination of ‘new’ races of Bremia lactucae inEurope by IBEB until 2002. In: Eucarpia leafy vegetables 2003.Proceedings of the Eucarpia Meeting on leafy vegetables genetics andbreeding. Noorwijkerhout, The Netherlands. Eds. Van Hintum T., LebedaA., Pink D., Schut J. pp 151-160; Van der Arend A J M, Gautier J,Grimault V, Kraan P, Van der Laan R, Mazet J, Michel H, Schut J W,Smilde D, De Witte I (2006) Identification and denomination of “new”races of Bremia lactucae in Europe by IBEB until 2006; incorporatedherein by reference; Plantum NL (Dutch association for breeding, tissueculture, production and trade of seeds and young plants), IBEB pressrelease, “New race of Bremia lactucae Bl:27 identified and nominated”,May 2010; Plantum NL (Dutch association for breeding, tissue culture,production and trade of seeds and young plants), IBEB press release,“New race of Bremia lactucae Bl:28 identified and nominated”, March2011; Plantum NL (Dutch association for breeding, tissue culture,production and trade of seeds and young plants), IBEB press release,“New races of Bremia lactucae, Bl:29, Bl:30 and Bl:31 identified andnominated”, August 2013; Plantum NL (Dutch association for breeding,tissue culture, production and trade of seeds and young plants), IBEBpress release, “A new race of Bremia lactucae, Bl:32 identified andnominated in Europe”, May 2015). Ca-I to Ca-VIII means Ca-I, Ca-IIA,Ca-IIB, Ca-III, Ca-IV (Schettini, T. M., Legg, E. J., Michelmore, R. W.,1991. Insensitivity to metalaxyl in California populations of Bremialactucae and resistance of California lettuce cultivars to downy mildew,Phytopathology 81(1). p. 64-′70), and Ca-V, Ca-VI, Ca-VII, Ca-VIII(Michelmore R. & Ochoa. O. “Breeding Crisphead Lettuce.” In: CaliforniaLettuce Research Board, Annual Report 2005-2006, 2006, Salinas, Calif.,pp. 55-68).

Resistance typically is tested by two interchangeable methods, describedby Bonnier, F. J. M. et al. (Euphytica, 61(3):203-211, 1992;incorporated herein by reference). One method involves inoculating 7-dayold seedlings, and observing sporulation 10 to 14 days later. The othermethod involves inoculating leaf discs with a diameter of 18 mm obtainedfrom a non-senescent, fully grown true leaf and observing sporulation 10days later.

As used herein, resistance against Nasonovia ribisnigri (Mosley), orcurrant-lettuce aphid, is defined as the plant characteristic whichresults in a non-feeding response of the aphid on the leaves of theplant in all stages between 5 true-leaf stage and harvestable plantstage (U.S. Pat. No. 5,977,443 to Jansen, J. P. A., “Aphid Resistance inComposites,” p. 12, 1999; incorporated herein by reference).

Resistance is tested by spreading at least ten aphids of biotype Nr:0 ona plant in a plant stage between 5 true leaves and harvestable stage,and observing the density of the aphid population on the plant as wellas the growth reduction after 14 days in a greenhouse, with temperaturesettings of 23 degrees Celsius in daytime and 21 degrees Celsius atnight. Day length is kept at 18 hours by assimilation lights.

As used herein, resistance against lettuce mosaic virus (LMV) may bedefined as the ability of the plant to grow normally after LMV infectionand to inhibit the virus transmission to seed. Resistance may be testedby mechanical inoculation of young plants in a climate cell orgreenhouse, as described by Pink, D. A. C. et al. (Plant Pathology,41(1): 5-12, 1992), incorporated herein by reference. Inoculatedresistant plants grow just as well as uninoculated plants and show nochlorosis or mosaic symptoms. The LMV isolate, which may be used fortesting, is Ls-1 (International Union for the Protection of NewVarieties of Plants [UPOV], Guidelines for the conduct of tests fordistinctness, uniformity and stability; 30 lettuce (Lactuca sativa L.),2002, p. 35; incorporated herein by reference).

As used herein, a plant with a non-heading plant habit is a plant whichdoes not make a head, i.e. with no head formation (International Unionfor the Protection of New Varieties of Plants [UPOV]), Guidelines forthe conduct of tests for distinctness, uniformity and stability; lettuce(Lactuca sativa L.) 2002; incorporated herein by reference). As areference, comparison variety Conversion RZ (81-01 RZ) has a non-headingplant habit. 81-296 RZ is also a non-heading lettuce of the Bataviatype.

As used herein, reduced wound-induced surface discoloration is definedas the ability of a lettuce plant, due to genetic information in itsgenome, to show a reduced or no discoloration of its wounded surface, ascompared to a control lettuce plant of the same type and same color notshowing the reduced wound-induced surface discoloration of its woundedsurface. For comparison of this trait, the standard variety ConversionRZ (81-01 RZ) can be used as a control plant for 81-296 RZ. As usedherein, the term “wound” is to mean the irreversible disturbance of thenatural plant, tissue and/or cell structure by methods like cutting,punching, slicing, abrasion, squashing, breaking, peeling, crushing,pressing, slashing, grinding, fluid injection, osmotic shock, detaching,mowing, shredding, rubbing and tearing.

This wound-induced surface discoloration trait can be tested by a leafdisc test in which a round leaf disc is punched from a fresh,fully-grown true leaf of a young plant, for example a plant at the 4-6true leaf stage. The leaf discs of the plant to be tested and thecontrol plant are incubated between two sheets of wetted filter paperfor about 3 to about 7 days at about 5° C. to 7° C. (such as, but notlimited to a 3 day incubation at about 5° C.), and the presence orabsence of pink discoloration around each leaf disc at the wound surfaceis observed. A leaf disc taken from a lettuce plant showing a reducedwound-induced surface discoloration trait due to genetic information inits genome which is responsible for the trait, will not show apink-colored discoloration (or only very faintly) at the edge of thewound surface, and is in any case less than the discoloration of a leafdisc taken from a control lettuce plant, for example a plant of thestandard variety Conversion RZ (81-01 RZ), not showing the reducedwound-induced surface discoloration trait which will show pink-coloreddiscoloration at the edge of the wound surface (For leaf disc test, seegenerally U.S. Pat. No. 8,809,631).

Embodiments of the inventions advantageously have one or more, and mostadvantageously all, of these characteristics.

In Table 2, the seed color, cotyledon shape and characteristics of thefourth leaf of 81-296 RZ are shown.

TABLE 2 Character 81-296 RZ Plant Type Batavia Seed Color WhiteCotyledon Shape Spatulate Shape of Fourth Leaf Elongated Rolling ofFourth Leaf Present Cupping of Fourth Leaf Uncupped Fourth Leaf ApicalMargin Moderately dentate Fourth Leaf Basal Margin Coarsely dentateUndulation Medium to slight Green color Dark green Anthocyanindistribution Absent

In Table 3, the mature leaf and head characteristics of 81-296 RZ areshown.

TABLE 3 Character 81-296 RZ Leaf Color Dark green (greyish) AnthocyaninAbsent Distribution Margin Incision Moderate Depth Margin IndentationShallowly dentate Undulations of the Strong Apical Margin Leaf SizeSmall Leaf Glossiness Moderate to dull Leaf Blistering Absent/slightLeaf Thickness Thick Trichomes Absent Plant size Small Head ShapeNon-heading Butt Shape Rounded Midrib Flattened

In Table 4, additional characteristic(s) of 81-296 RZ are shown.

TABLE 4 Character 81-296 RZ Wound-induced reduced surface discolorationResistance to B. lactucae Present races Bl:1 to Bl:32 and Ca-I toCa-VIII Resistance to N. Present ribisnigri, biotype Nr:0 Resistance toLMV, Present race LMV:1

In one aspect the invention provides a new lettuce (Lactuca sativa)variety of the Batavia type, designated 81-296 RZ. Lettuce cultivar81-296 RZ exhibits a combination of traits including resistance to downymildew (Bremia lactucae) races Bl:1 to Bl:32 and Ca-I to Ca-VIII,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,resistance to Lettuce Mosaic Virus (LMV) race LMV:1, as well as havingthick leaves, white colored seeds and a reduced wound-induced surfacediscoloration of the leaves.

In an embodiment, the invention relates to lettuce plants that have allthe physiological and morphological characteristics of the invention andhave acquired said characteristics by introduction of the geneticinformation that is responsible for the characteristics from a suitablesource, either by conventional breeding, or genetic modification, inparticular by cisgenesis or transgenesis. Cisgenesis is geneticmodification of plants with a natural gene, coding for an (agricultural)trait, from the crop plant itself or from a sexually compatible donorplant. Transgenesis is genetic modification of a plant with a gene froma non-crossable species or a synthetic gene.

Just as useful traits that may be introduced by backcrossing, usefultraits may be introduced directly into the plant of the invention, beinga plant of lettuce variety 81-296 RZ, by genetic transformationtechniques; and, such plants of lettuce variety 81-296 RZ that haveadditional genetic information introduced into the genome or thatexpress additional traits by having the DNA coding there for introducedinto the genome via transformation techniques, are within the ambit ofthe invention, as well as uses of such plants, and the making of suchplants.

Genetic transformation may therefore be used to insert a selectedtransgene into the plant of the invention, being a plant of lettucevariety 81-296 RZ or may, alternatively, be used for the preparation oftransgenes which may be introduced by backcrossing. Methods for thetransformation of plants, including lettuce, are well known to those ofskill in the art.

Vectors used for the transformation of lettuce cells are not limited solong as the vector may express an inserted DNA in the cells. Forexample, vectors which may comprise promoters for constitutive geneexpression in lettuce cells (e.g., cauliflower mosaic virus 35Spromoter) and promoters inducible by exogenous stimuli may be used.Examples of suitable vectors include pBI binary vector. The “lettucecell” into which the vector is to be introduced includes various formsof lettuce cells, such as cultured cell suspensions, protoplasts, leafsections, and callus. A vector may be introduced into lettuce cells byknown methods, such as the polyethylene glycol method, polycationmethod, electroporation, Agrobacterium-mediated transfer, particlebombardment and direct DNA uptake by protoplasts. To effecttransformation by electroporation, one may employ either friabletissues, such as a suspension culture of cells or embryogenic callus oralternatively one may transform immature embryos or other organizedtissue directly. In this technique, one would partially degrade the cellwalls of the chosen cells by exposing them to pectin-degrading enzymes(pectolyases) or mechanically wound tissues in a controlled manner.

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those which may be comprised oftungsten, platinum, and preferably, gold. For the bombardment, cells insuspension are concentrated on filters or solid culture medium.Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the macroprojectile stopping plate. Anillustrative embodiment of a method for delivering DNA into plant cellsby acceleration is the Biolistics Particle Delivery System, which may beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a surface covered with targetlettuce cells. The screen disperses the particles so that they are notdelivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectiles aggregate and maycontribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge. Microprojectile bombardment techniques are widely applicable, andmay be used to transform virtually any plant species, including a plantof lettuce variety 81-296 RZ.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA may be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast.Agrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations.Moreover, advances in vectors for Agrobacterium-mediated gene transferhave improved the arrangement of genes and restriction sites in thevectors to facilitate the construction of vectors capable of expressingvarious polypeptide coding genes. The vectors have convenientmulti-linker regions flanked by a promoter and a polyadenylation sitefor direct expression of inserted polypeptide coding genes.Additionally, Agrobacterium containing both armed and disarmed Ti genesmay be used for transformation. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene locustransfer. The use of Agrobacterium-mediated plant integrating vectors tointroduce DNA into plant cells, including lettuce plant cells, is wellknown in the art (See, e.g., U.S. Pat. Nos. 7,250,560 and 5,563,055).

Transformation of plant protoplasts also may be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments.

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for lettuce plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, a tandemly duplicated version of the CaMV 35Spromoter, the enhanced 35S promoter (P-e35S), the nopaline synthasepromoter, the octopine synthase promoter, the figwort mosaic virus(P-FMV) promoter (see U.S. Pat. No. 5,378,619), an enhanced version ofthe FMV promoter (P-eFMV) where the promoter sequence of P-FMV isduplicated in tandem, the cauliflower mosaic virus 19S promoter, asugarcane bacilliform virus promoter, a commelina yellow mottle viruspromoter, the promoter for the thylakoid membrane proteins (psaD, psaF,psaE, PC, FNR, atpC, atpD, cab, rbcS) (see U.S. Pat. No. 7,161,061), theCAB-1 promoter (see U.S. Pat. No. 7,663,027), the promoter from maizeprolamin seed storage protein (see U.S. Pat. No. 7,119,255), and otherplant DNA virus promoters known to express in plant cells. A variety ofplant gene promoters that are regulated in response to environmental,hormonal, chemical, and/or developmental signals may be used forexpression of an operably linked gene in plant cells, includingpromoters regulated by (1) heat, (2) light (e.g., pea rbcS-3A promoter,maize rbcS promoter, or chlorophyll a/b-binding protein promoter), (3)hormones, such as abscisic acid, (4) wounding (e.g., wunl, or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters.

Exemplary nucleic acids which may be introduced to the lettuce varietyof this invention include, for example, DNA sequences or genes fromanother species, or even genes or sequences which originate with or arepresent in lettuce species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA may include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a plant of lettuce variety 81-296 RZ.Non-limiting examples of particular genes and corresponding phenotypesone may choose to introduce into a lettuce plant include one or moregenes for insect tolerance, pest tolerance such as genes for fungaldisease control, herbicide tolerance, and genes for quality improvementssuch as yield, nutritional enhancements, environmental or stresstolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s).

Alternatively, the DNA coding sequences may affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms. The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product. Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention. (See also U.S. Pat. No.7,576,262, “Modified gene-silencing RNA and uses thereof.”)

U.S. Pat. Nos. 7,230,158, 7,122,720, 7,081,363, 6,734,341, 6,503,732,6,392,121, 6,087,560, 5,981,181, 5,977,060, 5,608,146, 5,516,667, eachof which, and all documents cited therein are hereby incorporated hereinby reference, consistent with the above INCORPORATION BY REFERENCEsection, are additionally cited as examples of U.S. Patents that mayconcern transformed lettuce and/or methods of transforming lettuce orlettuce plant cells, and techniques from these US Patents, as well aspromoters, vectors, etc., may be employed in the practice of thisinvention to introduce exogenous nucleic acid sequence(s) into a plantof lettuce variety 81-296 RZ (or cells thereof), and exemplify someexogenous nucleic acid sequence(s) which may be introduced into a plantof lettuce variety 81-296 RZ (or cells thereof) of the invention, aswell as techniques, promoters, vectors etc., to thereby obtain furtherplants of lettuce variety 81-296 RZ, plant parts and cells, seeds, otherpropagation material harvestable parts of these plants, etc. of theinvention, e.g. tissue culture, including a cell or protoplast, such asan embryo, meristem, cotyledon, pollen, leaf, anther, root, root tip,pistil, flower, seed or stalk.

The invention further relates to propagation material for producingplants of the invention. Such propagation material may comprise interalia seeds of the claimed plant and parts of the plant that are involvedin sexual reproduction. Such parts are for example selected from thegroup consisting of seeds, microspores, pollen, ovaries, ovules, embryosacs and egg cells. In addition, the invention relates to propagationmaterial which may comprise parts of the plant that are suitable forvegetative reproduction, for example cuttings, roots, stems, cells,protoplasts.

According to a further aspect thereof the propagation material of theinvention may comprise a tissue culture of the claimed plant. The tissueculture may comprise regenerable cells. Such tissue culture may bederived from leaves, pollen, embryos, cotyledon, hypocotyls,meristematic cells, roots, root tips, anthers, flowers, seeds and stems.(See generally U.S. Pat. No. 7,041,876 on lettuce being recognized as aplant that may be regenerated from cultured cells or tissue).

Also, the invention comprehends methods for producing a seed of a“81-296 RZ”-derived lettuce plant which may comprise (a) crossing aplant of lettuce variety 81-296 RZ, a sample of seed of which havingbeen deposited under NCIMB Accession No. 42738, with a second lettuceplant, and (b) whereby seed of a 81-296 RZ-derived lettuce plant form.Such a method may further comprise (c) crossing a plant grown from81-296 RZ-derived lettuce seed with itself or with a second lettuceplant to yield additional 81-296 RZ-derived lettuce seed, (d) growingthe additional 81-296 RZ-derived lettuce seed of step (c) to yieldadditional 81-296 RZ-derived lettuce plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to further generate 81-296 RZ-derived lettuce plants.

The invention further relates to the above methods that may furthercomprise selecting at steps b), d), and e), a 81-296 RZ-derived lettuceplant, exhibiting one or more or all of the physiological andmorphological characteristics of lettuce variety 81-296 RZ, a sample ofseed of said variety having been deposited under NCIMB accession No.42738, and other selected traits.

In particular, the invention relates to methods for producing a seed ofa 81-296 RZ-derived lettuce plant which may comprise (a) crossing aplant of lettuce variety 81-296 RZ, a sample of seed of which havingbeen deposited under NCIMB Accession No. 42738, with a second lettuceplant and (b) whereby seed of a 81-296 RZ-derived lettuce plant forms,wherein such a method may further comprise (c) crossing a plant grownfrom 81-296 RZ-derived lettuce seed with itself or with a second lettuceplant to yield additional 81-296 RZ-derived lettuce seed, (d) growingthe additional 81-296 RZ-derived lettuce seed of step (c) to yieldadditional 81-296 RZ-derived lettuce plants and selecting plantsexhibiting one or more or all of the physiological and morphologicalcharacteristics of lettuce variety 81-296 RZ, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to further generate 81-296 RZ-derived lettuce plants thatexhibit one or more or all of the physiological and morphologicalcharacteristics of lettuce variety 81-296 RZ.

The invention additionally provides a method of introducing at least onenew trait into a plant of lettuce variety 81-296 RZ which may comprise:(a) crossing a plant of lettuce variety 81-296 RZ, a sample of seed ofwhich having been deposited under NCIMB Accession No. 42738, with asecond lettuce plant that may comprise at least one new trait to produceprogeny seed; (b) harvesting and planting the progeny seed to produce atleast one progeny plant of a subsequent generation, wherein the progenyplant may comprise the at least one new trait; (c) crossing the selectedprogeny plant with a plant of lettuce variety 81-296 RZ, to producebackcross progeny seed; (d) harvesting and planting the backcrossprogeny seed to produce a backcross progeny plant, (e) repeating steps(c) and (d) for at least three additional generations to producebackcross progeny that may comprise the at least one new trait and allof the physiological and morphological characteristics of a plant oflettuce variety 81-296 RZ, when grown in the same environmentalconditions. The invention, of course, includes a lettuce plant producedby this method.

Backcrossing may also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This may be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred. When a plant of lettuce variety 81-296 RZ, a sampleof seed of which having been deposited under NCIMB Accession No. 42738,is used in backcrossing, offspring retaining one or more or all of thephysiological and morphological characteristics of lettuce variety81-296 RZ are progeny within the ambit of the invention. Backcrossingmethods may be used with the present invention to improve or introduce acharacteristic into a plant of the invention, being a plant of lettucevariety 81-296 RZ. See, e.g., U.S. Pat. No. 7,705,206 (incorporatedherein by reference consistent with the above INCORPORATION BY REFERENCEsection), for a general discussion relating to backcrossing.

The invention further involves a method of determining the genotype of aplant of lettuce variety 81-296 RZ, a sample of seed of which has beendeposited under NCIMB Accession No. 42738, or a first generation progenythereof, which may comprise obtaining a sample of nucleic acids fromsaid plant and detecting in said nucleic acids a plurality ofpolymorphisms. This method may additionally comprise the step of storingthe results of detecting the plurality of polymorphisms on a computerreadable medium. The plurality of polymorphisms are indicative of and/orgive rise to the expression of the physiological and morphologicalcharacteristics of lettuce variety 81-296 RZ.

There are various ways of obtaining genotype data from a nucleic acidsample. Genotype data may be gathered which is specific for certainphenotypic traits (e.g. gene sequences), but also patterns of randomgenetic variation may be obtained to construct a so-called DNAfingerprint. Depending on the technique used a fingerprint may beobtained that is unique for lettuce variety 81-296 RZ. Obtaining aunique DNA fingerprint depends on the genetic variation present in avariety and the sensitivity of the fingerprinting technique. A techniqueknown in the art to provide a good fingerprint profile is called AFLPfingerprinting technique (See generally U.S. Pat. No. 5,874,215), butthere are many other marker based techniques, such as RFLP (orRestriction fragment length polymorphism), SSLP (or Simple sequencelength polymorphism), RAPD (or Random amplification of polymorphic DNA)VNTR (or Variable number tandem repeat), Microsatellite polymorphism,SSR (or Simple sequence repeat), STR (or Short tandem repeat), SFP (orSingle feature polymorphism) DArT (or Diversity Arrays Technology), RADmarkers (or Restriction site associated DNA markers) (e.g. Baird et al.PloS One Vol. 3 e3376, 2008; Semagn et al. African Journal ofBiotechnology Vol. 5 number 25 pp. 2540-2568, 29 Dec. 2006). Nowadays,sequence-based methods are utilizing Single Nucleotide Polymorphisms(SNPs) that are randomly distributed across genomes, as a common toolfor genotyping (e.g. Elshire et al. PloS One Vol. 6: e19379, 2011;Poland et al. PloS One Vol. 7: e32253; Truong et al. PLoS One Vol. 7number 5: e37565, 2012).

With any of the aforementioned genotyping techniques, polymorphisms maybe detected when the genotype and/or sequence of the plant of interestis compared to the genotype and/or sequence of one or more referenceplants. As used herein, the genotype and/or sequence of a referenceplant may be derived from, but is not limited to, any one of thefollowing: parental lines, closely related plant varieties or species,complete genome sequence of a related plant variety or species, or thede novo assembled genome sequence of one or more related plant varietiesor species. For example, it is possible to detect polymorphisms for thecharacteristic of LMV resistance by comparing the genotype and/or thesequence of lettuce variety 81-296 RZ with the genotype and/or thesequence of one or more reference plants. The reference plant(s) usedfor comparison in this example may for example be, but is not limitedto, any of the comparison varieties Conversion RZ (81-01 RZ) or GrandRapids. The polymorphism revealed by these techniques may be used toestablish links between genotype and phenotype. The polymorphisms maythus be used to predict or identify certain phenotypic characteristics,individuals, or even species. The polymorphisms are generally calledmarkers. It is common practice for the skilled artisan to applymolecular DNA techniques for generating polymorphisms and creatingmarkers.

The polymorphisms of this invention may be provided in a variety ofmediums to facilitate use, e.g. a database or computer readable medium,which may also contain descriptive annotations in a form that allows askilled artisan to examine or query the polymorphisms and obtain usefulinformation.

As used herein “database” refers to any representation of retrievablecollected data including computer files such as text files, databasefiles, spreadsheet files and image files, printed tabulations andgraphical representations and combinations of digital and image datacollections. In a preferred aspect of the invention, “database” refersto a memory system that may store computer searchable information.

As used herein, “computer readable media” refers to any medium that maybe read and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard disc,storage medium and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM, DRAM, SRAM, SDRAM, ROM; and PROMs(EPROM, EEPROM, Flash EPROM), and hybrids of these categories such asmagnetic/optical storage media. A skilled artisan may readily appreciatehow any of the presently known computer readable mediums may be used tocreate a manufacture which may comprise computer readable medium havingrecorded thereon a polymorphism of the present invention.

As used herein, “recorded” refers to the result of a process for storinginformation in a retrievable database or computer readable medium. Forinstance, a skilled artisan may readily adopt any of the presently knownmethods for recording information on computer readable medium togenerate media which may comprise the polymorphisms of the presentinvention. A variety of data storage structures are available to askilled artisan for creating a computer readable medium where the choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats may be used to store the polymorphisms ofthe present invention on computer readable medium.

The present invention further provides systems, particularlycomputer-based systems, which contain the polymorphisms describedherein. Such systems are designed to identify the polymorphisms of thisinvention. As used herein, “a computer-based system” refers to thehardware, software and memory used to analyze the polymorphisms. Askilled artisan may readily appreciate that any one of the currentlyavailable computer-based system are suitable for use in the presentinvention.

Lettuce leaves are sold in packaged form, including without limitationas pre-packaged lettuce salad or as lettuce heads. Mention is made ofU.S. Pat. No. 5,523,136, incorporated herein by reference consistentwith the above INCORPORATION BY REFERENCE section, which providespackaging film, and packages from such packaging film, including suchpackaging containing leafy produce, and methods for making and usingsuch packaging film and packages, which are suitable for use with thelettuce leaves of the invention. Thus, the invention comprehends the useof and methods for making and using the leaves of the lettuce plant ofthe invention, as well as leaves of lettuce plants derived from theinvention. The invention further relates to a container which maycomprise one or more plants of the invention, or one or more lettuceplants derived from a plant of the invention, in a growth substrate forharvest of leaves from the plant in a domestic environment. This way theconsumer may pick very fresh leaves for use in salads. More generally,the invention includes one or more plants of the invention or one ormore plants derived from lettuce of the invention, wherein the plant isin a ready-to-harvest condition, including with the consumer picking hisown, and further including a container which may comprise one or more ofthese plants.

The invention is further described by the following numbered paragraphs:

1. A seed of lettuce variety 81-296 RZ, a sample of seed of said varietyhaving been deposited under NCIMB accession No. 42738.

2. A lettuce plant grown from the seed of paragraph 1.

3. The lettuce plant of paragraph 2, which is a plant grown from seedhaving been deposited under NCIMB accession No. 42738.

4. A lettuce plant, or a part thereof, having all the physiological andmorphological characteristics of the lettuce plant of paragraph 2.

5. A part of the plant of paragraph 2, wherein said part comprises amicrospore, pollen, ovary, ovule, embryo sac, egg cell, cutting, root,stem, cell or protoplast.

6. A tissue culture of regenerable cells or protoplasts from the lettuceplant of paragraph 2.

7. The tissue culture of paragraph 6, wherein said cells or protoplastsof the tissue culture are derived from a tissue comprising a leaf,pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip,anther, flower, seed or stem.

8. A lettuce plant regenerated from the tissue culture of paragraph 6 or7, wherein the regenerated plant expresses all of the physiological andmorphological characteristics of lettuce variety 81-296 RZ, a sample ofseed of said variety having been deposited under NCIMB accession No.42738.

9. A method of vegetatively propagating a plant of lettuce variety81-296 RZ comprising (a) collecting tissue capable of being propagatedfrom a plant of lettuce 81-296 RZ, a sample of seed of said varietyhaving been deposited under NCIMB accession No. 42738, (b) cultivatingthe tissue to obtain proliferated shoots and rooting the proliferatedshoots to obtain rooted plantlets, and (c) optionally growing plantsfrom the rooted plantlets.

10. A method for producing a progeny plant of lettuce cultivar 81-296RZ, comprising crossing a plant designated 81-296 RZ with itself or withanother lettuce plant, harvesting the resultant seed, and growing saidseed.

11. A progeny plant produced by the method of paragraph 10, wherein saidprogeny plant exhibits a combination of traits including resistance todowny mildew (Bremia lactucae) races Bl:1 to Bl:32 and Ca-I to Ca-VIII,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,resistance to Lettuce Mosaic Virus (LMV) race LMV:1, as well as havingthick leaves, white colored seeds and a reduced wound-induced surfacediscoloration of the leaves.

12. A progeny plant produced by the method of paragraph 10, wherein saidprogeny exhibits all the morphological and physiological characteristicsof the lettuce variety designated 81-296 RZ, a sample of seed of saidvariety having been deposited under NCIMB accession No. 42738.

13. The progeny plant of paragraph 11, wherein said progeny plant ismodified in one or more other characteristics.

14. The progeny plant of paragraph 13, wherein the modification iseffected by mutagenesis.

15. The progeny plant of paragraph 13, wherein the modification iseffected by transformation with a transgene.

16. A method of producing a lettuce seed comprising crossing a maleparent lettuce plant with a female parent lettuce plant and harvestingthe resultant lettuce seed, wherein said male parent lettuce plant orsaid female parent lettuce plant is the lettuce plant of paragraph 2.

17. A lettuce seed produced by the method of paragraph 16.

18. A lettuce plant produced by growing the seed of paragraph 17.

19. A method for producing a seed of a 81-296 RZ-derived lettuce plantcomprising (a) crossing a plant of lettuce variety 81-296 RZ, a sampleof seed of which having been deposited under NCIMB Accession No. 42738,with a second lettuce plant, and (b) whereby seed of a 81-296 RZ-derivedlettuce plant forms.

20. The method of paragraph 19 further comprising (c) crossing a plantgrown from 81-296 RZ-derived lettuce seed with itself or with a secondlettuce plant to yield additional 81-296 RZ-derived lettuce seed, (d)growing the additional 81-296 RZ-derived lettuce seed of step (c) toyield additional 81-296 RZ-derived lettuce plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to generate further 81-296 RZ-derived lettuce plants, and(f) whereby seed of a 81-296 RZ-derived lettuce plant form.

21. A seed produced by the method of paragraph 19.

22. A plant grown from the seed produced by the method of paragraph 21.

23. A method of introducing at least one new trait into a plant oflettuce variety 81-296 RZ comprising: (a) crossing a plant of lettucevariety 81-296 RZ, a sample of seed of which having been deposited underNCIMB Accession No. 42738, with a second lettuce plant that comprises atleast one new trait to produce progeny seed, (b) harvesting and plantingthe progeny seed to produce at least one progeny plant of a subsequentgeneration, wherein the progeny plant comprises the at least one newtrait, (c) crossing the progeny plant with a plant of lettuce variety81-296 RZ to produce backcross progeny seed, (d) harvesting and plantingthe backcross progeny seed to produce a backcross progeny plant, and (e)repeating steps (c) and (d) for at least three additional generations toproduce a lettuce plant of variety 81-296 RZ comprising at least one newtrait and all of the physiological and morphological characteristics ofa plant of lettuce variety 81-296 RZ, when grown in the sameenvironmental conditions.

24. A method of producing a plant of lettuce variety 81-296 RZcomprising at least one new trait, the method comprising introducing amutation or transgene conferring the at least one new trait into a plantof lettuce variety 81-296 RZ, wherein a sample of seed of said varietyhas been deposited under NCIMB Accession No. 42738.

25. The lettuce plant produced by the method of paragraph 23.

26. A method for producing lettuce leaves as a food product comprisingsowing the seed of a paragraph 1 and growing the seed into a harvestablelettuce plant and harvesting the head or leaves of said plant.

27. A method for producing lettuce leaves as a fresh vegetablecomprising packaging leaves of a plant of paragraph 2.

28. A method for producing lettuce leaves as a processed food comprisingprocessing leaves of a plant of paragraph 2.

29. A container comprising one or more lettuce plants of paragraph 2 forharvest of leaves.

30. A method of determining the genotype of a plant of lettuce variety81-296 RZ, a sample of seed of which has been deposited under NCIMBAccession No. 42738, or a first generation progeny thereof, comprisingobtaining a sample of nucleic acids from said plant and comparing saidnucleic acids to a sample of nucleic acids obtained from a referenceplant, and detecting a plurality of polymorphisms between the twonucleic acid samples, wherein the plurality of polymorphisms areindicative of lettuce variety 81-296 RZ and/or give rise to theexpression of any one or more, or all, of the physiological andmorphological characteristics of lettuce variety 81-296 RZ of paragraph2.

31. A lettuce plant produced by the method of paragraph 24.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention is not to belimited to particular details set forth in the above description as manyapparent variations thereof are possible without departing from thespirit or scope of the present invention.

What is claimed is:
 1. A seed of lettuce variety 81-296 RZ, a sample ofseed of said variety having been deposited under NCIMB Accession No.42738.
 2. A lettuce plant grown from the seed of claim
 1. 3. A lettuceplant, or a part thereof, having all the physiological and morphologicalcharacteristics of the lettuce plant of claim
 2. 4. A part of the plantof claim 2, wherein said part comprises a microspore, pollen, ovary,ovule, embryo sac, egg cell, cutting, root, stem, cell or protoplast. 5.A tissue culture of regenerable cells or protoplasts from the lettuceplant of claim
 2. 6. The tissue culture as claimed in claim 5, whereinsaid cells or protoplasts of the tissue culture are derived from atissue comprising a leaf, pollen, embryo, cotyledon, hypocotyl,meristematic cell, root, root tip, anther, flower, seed or stem.
 7. Alettuce plant regenerated from the tissue culture of claim 5, whereinthe regenerated plant expresses all of the physiological andmorphological characteristics of lettuce variety 81-296 RZ, a sample ofseed of said variety having been deposited under NCIMB accession No.42738.
 8. A method of vegetatively propagating a plant of lettucevariety 81-296 RZ comprising (a) collecting tissue capable of beingpropagated from a plant of lettuce 81-296 RZ, a sample of seed of saidvariety having been deposited under NCIMB Accession No. 42738, (b)cultivating the tissue to obtain proliferated shoots and rooting theproliferated shoots to obtain rooted plantlets, and (c) optionallygrowing plants from the rooted plantlets.
 9. A method for producing aprogeny plant of lettuce cultivar 81-296 RZ, comprising crossing a plantdesignated 81-296 RZ, representative seed which having been depositedunder NCIMB Accession No. 42738, with itself or with another lettuceplant, harvesting the resultant seed, and growing said seed.
 10. Aprogeny plant produced by the method of claim 9, wherein said progenyplant exhibits all of the morphological and physiologicalcharacteristics of a lettuce plant, representative seed of which havingbeen deposited under NCIMB Accession No.
 42738. 11. A method ofproducing a lettuce seed comprising crossing a male parent lettuce plantwith a female parent lettuce plant and harvesting the resultant lettuceseed, wherein said male parent lettuce plant or said female parentlettuce plant is the lettuce plant of claim
 2. 12. An F1 lettuce seedproduced by the method of claim
 11. 13. A lettuce plant produced bygrowing the seed of claim
 12. 14. A method for producing a seed of a81-296 RZ-derived lettuce plant comprising (a) crossing a plant oflettuce variety 81-296 RZ, a sample of seed of which having beendeposited under NCIMB Accession No. 42738, with a second lettuce plant,and (b) whereby seed of a 81-296 RZ-derived lettuce plant forms.
 15. Themethod of claim 14 further comprising (c) crossing a plant grown from81-296 RZ-derived lettuce seed with itself or with a second lettuceplant to yield additional 81-296 RZ-derived lettuce seed, (d) growingthe additional 81-296 RZ-derived lettuce seed of step (c) to yieldadditional 81-296 RZ-derived lettuce plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to generate further 81-296 RZ-derived lettuce plants, and(f) whereby seed of a 81-296 RZ-derived lettuce plant forms.
 16. Amethod of introducing at least one new trait into a plant of lettucevariety 81-296 RZ comprising: (a) crossing a plant of lettuce variety81-296 RZ, a sample of seed of which having been deposited under NCIMBAccession No. 42738, with a second lettuce plant that comprises at leastone new trait to produce progeny seed, (b) harvesting and planting theprogeny seed to produce at least one progeny plant of a subsequentgeneration, wherein the progeny plant comprises the at least one newtrait, (c) crossing the progeny plant with a plant of lettuce variety81-296 RZ to produce backcross progeny seed, (d) harvesting and plantingthe backcross progeny seed to produce a backcross progeny plant, and (e)repeating steps (c) and (d) for at least three additional generations toproduce a lettuce plant of variety 81-296 RZ comprising at least one newtrait and otherwise all of the physiological and morphologicalcharacteristics of a plant of lettuce variety 81-296 RZ, when grown inthe same environmental conditions.
 17. A method of producing a plant oflettuce variety 81-296 RZ comprising at least one new trait, the methodcomprising introducing a mutation or transgene conferring the at leastone new trait into a plant of lettuce variety 81-296 RZ, wherein asample of seed of said variety has been deposited under NCIMB AccessionNo.
 42738. 18. A lettuce plant produced by the method of claim 16,wherein said plant comprises the at least one new trait and otherwiseall of the morphological and physiological characteristics of a plant oflettuce variety 81-296 RZ.
 19. A method for producing lettuce leaves asa food product comprising sowing the seed of claim 1 and growing theseed into a harvestable lettuce plant and harvesting the head or leavesof said plant.
 20. A method for producing lettuce leaves as a freshvegetable comprising packaging leaves of the plant of claim
 2. 21. Amethod for producing lettuce leaves as a processed food comprisingprocessing leaves of the plant of claim
 2. 22. A container comprisingone or more lettuce plants of claim 2 for harvest of leaves.
 23. Amethod of determining the genotype of a plant of lettuce variety 81-296RZ, a sample of seed of which has been deposited under NCIMB AccessionNo. 42738, comprising obtaining a sample of nucleic acids from saidplant and comparing said nucleic acids to a sample of nucleic acidsobtained from a reference plant, and detecting a plurality ofpolymorphisms between the two nucleic acid samples, wherein theplurality of polymorphisms are indicative of lettuce variety 81-296 RZand/or give rise to the expression of any one or more, or all, of thephysiological and morphological characteristics of lettuce variety81-296 RZ as claimed in claim
 2. 24. A method for producing a modifiedlettuce plant comprising mutagenizing the seed of claim
 1. 25. A lettuceplant designated 81-296 RZ, representative seed of which having beendeposited under NCIMB Accession No. 42738, further comprising atransgene.
 26. A method for producing a modified lettuce plantcomprising mutagenizing the plant of claim
 2. 27. A method for producinga modified lettuce plant comprising mutagenizing the plant of claim 3.28. A method for producing a modified lettuce plant comprisingmutagenizing the plant of claim
 7. 29. A method for producing a modifiedlettuce plant comprising mutagenizing the plant part of claim
 3. 30. Amethod for producing a modified lettuce plant comprising mutagenizingthe plant part of claim
 4. 31. A method for producing a modified lettuceplant comprising mutagenizing the tissue culture of claim
 5. 32. Theplant of claim 25, wherein the transgene is introduced viatransformation.